PROJECT SUMMARY AND ABSTRACT The RAS genes KRAS, NRAS, or HRAS, are commonly mutated in human cancers. Clinically inhibiting RAS has proven challenging and RAS-mutant cancers remain some of the most intractable diseases, even to immunotherapies. It is thus critical to elucidate oncogenic RAS signaling, not only to better understand the tumorigenic process, but also to identify new potential therapeutic targets. To this end, I exploited the novel technique of BirA-mediated proximity labeling to identify proteins within the immediate vicinity (interactome) of each RAS isoform. I then screened an sgRNA library targeting interactome components for genes promoting RAS transformed cell growth, identifying the druggable phosphatidylinositol phosphate lipid kinase PIP5K1A as specifically driving KRAS oncogenesis. PIP5K1A represents an entirely new therapeutic target in KRAS-mutant cancers, and suggests that other proteins in the RAS interactome may similarly mediate RAS oncogenesis. I will capitalized on these discoveries in three aims. As PIP5K1A is a druggable kinase it provides a way to specifically inhibit KRAS oncogenesis, which could be exploited to enhance the antineoplastic activity of drugs targeting RAS effector pathways. Thus, in aim 1 I will elucidate the role and therapeutic potential of targeting PIP5K1A in KRAS-mutant cancers. The identification of PIP5K1A promoting KRAS oncogenesis suggests that other interactome proteins may similarly mediate RAS function. Thus, in aim 2 I will mine the RAS interactome for novel modifiers of RAS oncogenesis, focusing on the interactome protein EFR3A as a potential general mediator of oncogenic RAS-driven tumorigenesis. Finally, the RAS interactome is most certainly dynamic, varying under different conditions. Determining the content of the RAS interactome under distinct settings may thus identify new vulnerabilities specific to diverse cellular conditions. Thus, in aim 3 I will probe the RAS interactome in response to cellular perturbations. In sum, I will expand upon my discovery that PIP5K1A promotes KRAS oncogenesis to explore this kinase as a new therapeutic target and identify other novel therapeutic vulnerabilities that exists within the RAS interactome. The K99 segment of this grant will complete my training in RAS signal transduction, extend my training into phosphoproteomics, xenograft and genetically engineered mouse models of tumorigenesis. The R00 segment will capitalize on the use of proximity labeling to study the dynamic nature of oncogenic RAS signaling. My long-term goal is to transition into an independent investigator and apply systems biology approaches to uncover the signaling circuitry of oncogene drivers with the objective of identifying novel therapeutic vulnerabilities in RAS-mutant cancers.